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ORIGINAL RESEARCHpublished: 09 January 2017

doi: 10.3389/fmicb.2016.02144

Edited by:Martin G. Klotz,

City University of New York, USA

Reviewed by:Barry Scott,

Massey University, New ZealandFabienne Malagnac,

Université Paris Sud, France

*Correspondence:Lena Studt

lena.studt@boku.ac.atJoseph Strauss

joseph.strauss@boku.ac.at

Specialty section:This article was submitted toFungi and Their Interactions,

a section of the journalFrontiers in Microbiology

Received: 08 September 2016Accepted: 20 December 2016

Published: 09 January 2017

Citation:Studt L, Janevska S, Arndt B,

Boedi S, Sulyok M, Humpf H-U,Tudzynski B and Strauss J (2017)

Lack of the COMPASS ComponentCcl1 Reduces H3K4 Trimethylation

Levels and Affects Transcriptionof Secondary Metabolite Genes

in Two Plant–Pathogenic FusariumSpecies. Front. Microbiol. 7:2144.

doi: 10.3389/fmicb.2016.02144

Lack of the COMPASS ComponentCcl1 Reduces H3K4 TrimethylationLevels and Affects Transcription ofSecondary Metabolite Genes in TwoPlant–Pathogenic Fusarium SpeciesLena Studt1,2*, Slavica Janevska2, Birgit Arndt3, Stefan Boedi1, Michael Sulyok4,Hans-Ulrich Humpf3, Bettina Tudzynski2 and Joseph Strauss1*

1 Division of Microbial Genetics and Pathogen Interactions, Department of Applied Genetics and Cell Biology,BOKU-University of Natural Resources and Life Sciences, Vienna, Tulln an der Donau, Austria, 2 Institute for Plant Biologyand Biotechnology, Westfälische Wilhelms University, Münster, Germany, 3 Institute of Food Chemistry, WestfälischeWilhelms University, Münster, Germany, 4 Center for Analytical Chemistry, Department IFA-Tulln, BOKU-University of NaturalResources and Life Sciences, Vienna, Tulln an der Donau, Austria

In the two fungal pathogens Fusarium fujikuroi and Fusarium graminearum, secondarymetabolites (SMs) are fitness and virulence factors and there is compelling evidencethat the coordination of SM gene expression is under epigenetic control. Here, wecharacterized Ccl1, a subunit of the COMPASS complex responsible for methylatinglysine 4 of histone H3 (H3K4me). We show that Ccl1 is not essential for viability but aregulator of genome-wide trimethylation of H3K4 (H3K4me3). Although, recent work inFusarium and Aspergillus spp. detected only sporadic H3K4 methylation at the majorityof the SM gene clusters, we show here that SM profiles in CCL1 deletion mutants arestrongly deviating from the wild type. Cross-complementation experiments indicate highfunctional conservation of Ccl1 as phenotypes of the respective 4ccl1 were rescued inboth fungi. Strikingly, biosynthesis of the species-specific virulence factors gibberellicacid and deoxynivalenol produced by F. fujikuroi and F. graminearum, respectively,was reduced in axenic cultures but virulence was not attenuated in these mutants, aphenotype which goes in line with restored virulence factor production levels in planta.This suggests that yet unknown plant-derived signals are able to compensate for Ccl1function during pathogenesis.

Keywords: Fusarium, H3K4 methylation, COMPASS, secondary metabolism, virulence, histone modification

INTRODUCTION

Filamentous fungi produce a huge arsenal of secondary metabolites (SMs), small molecular-weightcompounds that are not needed for the survival of the fungus but are thought to contribute tothe fitness of its producer in some way (Fox and Howlett, 2008; Reverberi et al., 2010; Rohlfs andChurchill, 2011). Among those SMs are potent toxins that frequently contaminate food and feed(Streit et al., 2013), but also pharmaceuticals and plant hormones that are applied in medicine andagriculture, respectively (Dreyfuss et al., 1976; Anke et al., 1977; Rademacher, 1997; Manzoni andRollini, 2002). SM production is energy consuming, and thus, only initiated when advantageous for

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the producer. This implies a tight regulatory network functioningin induction or repression of the respective SM genes. Genesinvolved in SM biosynthesis are generally clustered (Keller andHohn, 1997), and thus, prone to chromatin level regulation.The chromatin landscape is either loosely packed, open fortranscription (euchromatin) or densely packed and silenced(heterochromatin). Both chromatin states are associated withthe activity of certain histone-modifying enzymes that are oftenpart of large enzyme complexes and involved in the addition orremoval of residues at defined amino acids of the histone proteintails (e.g., Bannister and Kouzarides, 2011; Gacek and Strauss,2012).

One such complex is COMPASS (complex of proteinsassociated with Set1) that is highly conserved from yeastto humans and plants (Miller et al., 2001; Krogan et al.,2002; Hughes et al., 2004; Lee et al., 2007; Wu et al., 2008;Jiang et al., 2011; Shilatifard, 2012). The catalytic subunit,the methyltransferase Set1 (Kmt2), was first identified and co-purified in Saccharomyces cerevisiae together with a complexconsisting of eight subunits, i.e., Set1, Bre2, Swd1, Spp1, Swd2,Swd3, Sdc1, and Shg1 (Nislow et al., 1997; Miller et al., 2001;Roguev et al., 2001). Set1 catalyzes mono-, di-, and trimethylationof the fourth lysine at histone 3 (H3K4me1/2/3) (Roguev et al.,2001; Krogan et al., 2002; Liu et al., 2015). However, Set1 aloneis not functional as each of the subunits has a specific functionin complex assembly and integrity, pattern of global H3K4methylation and distribution of H3K4me along active genes(Dehé et al., 2006; Shilatifard, 2012). One member of COMPASSis Bre2 (Ash2 in Schizosaccharomyces pombe and Magnaportheoryzae, CclA in Aspergillus spp.) that is specifically involvedin H3K4me3 with limited to no effect on di- and no effecton monomethylation (Schlichter and Cairns, 2005; Schneideret al., 2005; Dehé et al., 2006; Bok et al., 2009; Palmer et al.,2013; Mikheyeva et al., 2014; Pham et al., 2015). Notably, whileH3K4 methylation is generally associated with transcriptionalgene activation, COMPASS-mediated silencing of genes locatednear chromosome telomeres has also been described (Briggs et al.,2001; Krogan et al., 2002; Bok et al., 2009; Mikheyeva et al., 2014).Deletion of the bre2 homolog cclA in Aspergillus spp. resulted inincreased expression of several SM cluster genes (Bok et al., 2009;Palmer et al., 2013; Shinohara et al., 2016).

In this study, we analyzed the influence of the bre2homolog CCL1 in two Fusarium spp., that is the rice pathogenFusarium fujikuroi (teleomorph Gibberella fujikuroi) responsiblefor bakanae disease on rice (Wiemann et al., 2013), and the cerealcrop pathogen Fusarium graminearum (teleomorph Gibberellazeae) causing severe epidemics of head blight (Kazan et al., 2012).These two fungi are able to produce numerous SMs. Notably mostof the SMs are species-specific and produced by only one of thetwo fusaria. Prominent examples are the natural plant hormonegibberellins (GAs) and the mycotoxin deoxynivalenol (DON)produced by F. fujikuroi and F. graminearum, respectively, whichare both directly associated with the disease symptoms caused bythese two fungi (Proctor et al., 1995; Jansen et al., 2005; Wiemannet al., 2013). In addition, F. fujikuroi and F. graminearum producesimilar but distinct polyketide synthase (PKS)-derived pigmentsthat are thought to function as protectants against environmental

stress, i.e., the mycelial pigments bikaverin (BIK) and aurofusarin(AUR), respectively (Malz et al., 2005; Wiemann et al., 2009).Several additional SMs have been characterized in both fungiover the last years. Those include distinct SMs produced by onlyone of the two fusaria such as fusaric acid (FSA), fumonisins,fujikurins, or apicidin F produced by F. fujikuroi (von Bargenet al., 2013, 2015; Niehaus et al., 2014a; Rösler et al., 2016),and zearalenone (ZON), fusarielins, culmorin, or butenolideproduced by F. graminearum (Hansen et al., 2012; Sørensen et al.,2012). There are also common SMs, such as the fusarins (FUS), afamily of structurally related polyketide mycotoxins (Gelderblomet al., 1984; Díaz-Sánchez et al., 2012; Niehaus et al., 2013),and some non-ribosomal peptide synthetase (NRPS)-derivedsiderophores (Hansen et al., 2015). In this study, we show thatthe COMPASS component-encoding gene CCL1 is a conservedyet not essential gene in the two Fusarium spp. analyzed, but thatdeletion had a drastic effect on the SM profiles.

MATERIALS AND METHODS

Fungal Strains, Media, and GrowthConditionsThe wild type strain F. fujikuroi IMI58289 (CommonwealthMycological Institute, Kew, UK) as well as the F. graminearumwild type strain PH-1 (FGSC 9075, NRRL 31084) were usedas parental strains for knock-out experiments. For SM analysisand ChIP experiments, F. fujikuroi strains were pre-incubatedfor 72 h in 300 mL Erlenmeyer flasks with 100 mL Darkenmedium (DVK) (Darken et al., 1959) on a rotary shaker inconstant darkness at 180 rpm and 28◦C. A 500 µL aliquotof this culture was used for inoculation of synthetic ICI(Imperial Chemical Industries, Ltd, UK) medium (Geissmanet al., 1966), with varying nitrogen sources, either 6 mM (nitrogenstarvation) or 60 mM (nitrogen surplus) glutamine. Incubationwas carried on for additional three to 7 days in case of chromatinimmunoprecipitation (ChIP) analyses and chemical analyses,respectively. F. graminearum strains were grown for 2 weekson solid potato dextrose agar (PDA; Sigma-Aldrich, Germany)for SM quantification. For gene expression analysis, the wildtype strain was grown for up to 2 weeks on solid PDA andfor ChIP analysis, strains were grown for 4 days on PDA. Forprotoplasting of F. fujikuroi, 500 µL of the pre-incubated culturewas taken and transferred into 100 mL ICI medium with 10 g/Lfructose instead of glucose and 1 g/L (NH4)2SO4 as nitrogensource and incubated on a rotary shaker at 28◦C and 180 rpmno longer than 16 h. For protoplasting of F. graminearum,YPG medium (Brown et al., 2002) was inoculated with ∼106

conidia and incubated overnight on a rotary shaker at 24◦C and180 rpm. For conidiation, F. graminearum strains were grownfor 7 days in liquid carboxymethylcellulose media (CMC) on arotary shaker at 24◦C and 180 rpm (Cappellini and Peterson,1965). In case of F. fujikuroi, solidified vegetable juice (V8)medium was inoculated with 5 mm agar plaques and incubatedfor 7 days at room temperature. For DNA isolation and westernblot analysis, the fungal strains were grown on solidified completemedium (CM) (Pontecorvo et al., 1953) covered with cellophane

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sheets for 3 days at 28◦C in case of F. fujikuroi or 20◦C in caseof F. graminearum. Hyphal growth of both fungi was assessedon solid CM as rich media, while Fusarium minimal medium(FMM) (Reyes-Dominguez et al., 2012) was chosen as a minimalmedium. Plates were incubated for 5 days at 28 and 20◦C in caseof F. fujikuroi and F. graminearum, respectively.

Plasmid ConstructionsPlasmids for F. fujikuroi and F. graminearum knock-out,complementation and cross-complementation strains weregenerated using yeast recombinational cloning (Colot et al.,2006). All primers used for polymerase chain reaction (PCR)were obtained from Eurofins GmbH (Ebersberg, Germany).For knock-out constructs, the upstream (5′) and downstream(3′) sequences of FfCCL1 and FgCCL1 were amplified fromF. fujikuroi IMI58289 and F. graminearum PH-1 genomic DNA,respectively, with the following primer pairs: FfCCL1_5F andFfCCL1_5R for upstream and FfCCL1_3F and FfCCL1_3R fordownstream regions in case of F. fujikuroi and FgCCL1_5Fand FgCCL1_5R for upstream and FgCCL1_3F and FgCCL1_3Rfor downstream regions in case of F. graminearum. In bothcases, hygromycin B was used as resistance marker. Thehygromycin B resistance cassette, consisting of the hygromycinB phosphotransferase gene hph (Gritz and Davies, 1983), drivenby the trpC promoter, was amplified using the primer pairhph-F and hph-R from the template pCSN44 (Staben et al.,1989). The S. cerevisiae strain FY834 was transformed withthe obtained fragments (Winston et al., 1995), as well aswith the EcoRI/XhoI restricted plasmid pRS426 (Christiansonet al., 1992) yielding plasmids p1ffccl1 and p1fgccl1. Forcomplementation of 1ffccl1, the respective gene including about1 kb of the upstream region was amplified using a proof-readingpolymerase and the primer pair FfCCL1_5F and FfCCL1C_IL_R.The gene was fused to the glucanase terminator sequence,BcTgluc, obtained from Botrytis cinerea B05.10 using a proof-reading polymerase and the primer pair BcGlu-term-F2 andTgluc-nat1-R, followed by the nourseothricin resistance cassette,natR, itself driven by the trpC promoter. The nourseothricinresistance cassette was amplified with the primer pair Hph-R-nat1 and Hph-F using a proof-reading polymerase and thetemplate pZPnat1 (GenBank accession no. AY631958.1). Thedownstream region of FfCCL1 was amplified using the sameprimer pair as in the deletion approach, i.e., FfCCL1_3F andFfCCL1_3R. S. cerevisiae FY834 was transformed with theobtained fragments as well as with the EcoRI/XhoI restrictedpRS426 yielding plasmid pFfCCL1Cil (Supplementary FigureS1A). Similarly, complementation of 1fgccl1 was accomplishedby amplification of FgCCL1 and about 1 kb upstream region usingthe primer pair FgCCL1_IL_F and FgCCL1_Tgluc_IL_R as wellas a proof-reading polymerase. The gene was fused to the BcTglucterminator sequence followed by the geniticin resistance cassette,genR, which was amplified from pKS-Gen (Bluhm et al., 2008)using a proof-reading polymerase and the primer pair Geni-F and Geni-Tgluc-R. The downstream region of FgCCL1 wasamplified from F. graminearum PH-1 genomic DNA using theprimer pair FgCCL1_3F and FgCCL1_3R. S. cerevisiae FY834was transformed with the obtained fragments as well as with

the EcoRI/XhoI restricted pRS426 yielding plasmid pFgCCL1Cil

(Supplementary Figure S1A).For cross-complementation of FgCCL1 in 1ffccl1, FgCCL1

was amplified from F. graminearum PH-1 genomic DNA usinga proof-reading polymerase with the primer pair FgCCL1-FfCCL1_F and FgCCL1-IL_TglucR. The gene was drivenby the FfCCL1 native promoter that was amplified fromF. fujikuroi IMI58289 genomic DNA using the primer pairFfCCL1_5F and FfCCL1_5R2, and fused to BcTgluc followedby the nourseothricin resistance cassette, nat1R, as describedearlier. Downstream region was amplified from F. fujikuroiIMI58289 genomic DNA using the primer pair FfCCL1_3Fand FfCCL1_3R. S. cerevisiae FY834 was transformed with theobtained fragments as well as with the EcoRI/XhoI restrictedpRS426 yielding plasmid p1ffccl1/FgCCL1 (SupplementaryFigure S1B). For cross-complementation of FfCCL1 in 1fgccl1,the F. fujikuroi FfCCL1 gene was amplified from F. fujikuroiIMI58289 genomic DNA with the primer pair FfCCL1-IL_Fand FfCCL1-Tgluc-IL_R using a proof-reading polymerase.The gene was fused to BcTgluc followed by the geneticinresistance cassette, genR. Upstream and downstream regionswere amplified using the primer pairs FgCCL1_5F and FgCCL1-FfCCL1_5R and FgCCL1_3F and FgCCL1_3R, respectively.S. cerevisiae FY834 was transformed with the obtainedfragments as well as with the EcoRI/XhoI restricted pRS426yielding plasmid p1fgccl1/FfCCL1 (Supplementary FigureS1B). Correct assembly of the resulting vectors, pFfCCL1Cil,pFgCCL1Cil, p1ffccl1/FgCCL1 and p1fgccl1/FfCCL1 was verifiedby sequencing.

Fungal TransformationsFor generation of F. fujikuroi mutants, protoplasts were preparedfrom the wild type strain IMI58289. Transformation was carriedout as previously described (Tudzynski et al., 1996). About107 protoplasts were transformed with about 10 µg of theamplified replacement cassettes for generating the knock-outs.The knock-out fragments were amplified prior to transformationusing p1ffccl1, the primer pair FfCCL1_5F and FfCCL1_5R andTAKARA polymerase. In case of F. graminearum, we used thesplit-marker approach (Goswami, 2012). For this, two knock-out fragments containing about two thirds of hph each wereamplified from p1fgccl1 using the primer pairs FgCCL1_5F andsplit-mark hph_F as well as split-mark hph_R and FgCCL1_3R.Transformation of F. graminearum was according to F. fujikuroiwith minor adjustments. For complementation and cross-complementation of 1ffccl1, the deletion mutant 1ffccl1_T9 wastransformed with 10 µg of the respective plasmid pFfCCL1Cil

and p1ffccl1/FgCCL1. Plasmids were linearized with either PvuIIor SspI prior to transformation (Supplementary Figure S1).Complementation and cross-complementation of 1fgccl1 wasperformed according to the split-marker approach (Goswami,2012). Therefore, knock-out fragments were amplified with thefollowing primer pairs: FgCCL1_5F and genR_splitF as well asgenR_splitR and FgCCL1_3R. All transformed protoplasts wereregenerated as described (Tudzynski et al., 1999). The mediumcontained 100 ppm of the appropriate resistance marker. Singleconidial cultures were established from either hygromycin B-,

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nourseothricin-, or geneticin-resistant transformants and usedfor subsequent DNA isolation. In case of 1ffccl1 and 1fgccl1mutants, homologous integration of the resistance cassette wasverified using the primer pairs dia_FfCCL1_5′ and TrpC-T forthe upstream part as well as dia_FfCCL1_3′ and TrpC-P forthe downstream part in case of F. fujikuroi and dia_FgCCL1_5′and TrpC-T (upstream) as well as dia_FgCCL1_3′ and TrpC-P(downstream) in case of F. graminearum. Absence of the wildtype gene was verified using the primer pairs FfCCL1_WT-F andFfCCL1_WT-R as well as FgCCL1_WT_F and FgCCL1_WT_Rin case of F. fujikuroi and F. graminearum, respectively(Supplementary Figure S2). Homologous integration of theF. fujikuroi (cross-) complementation constructs was verifiedusing the primer pairs dia_FfCCL1_5′ and FfCCL1_Seq1(complementation), dia_FfCCL1_5′ and FgCCL1_IL_dia(cross-complementation) for the upstream parts as wellas dia_FfCCL1_3′//Nat1_R1 for the downstream parts.Accordingly, homologous integration in F. graminearumwas verified using the primer pairs dia_ILCom_FgCCL1_5Fand dia_ILCom_FgCCL1_5R (complementation) as well asdia_ILCom_FgCCL1_5F and dia_IL-CCom_FgxFfCCL1_5R(cross-complementation) for the upstream region as well as gen-Seq3 and dia_IL-Com_FgCCL1_3R for the downstream region.Absence of hph was checked by phenotypic characterizationof the complemented mutants, thus, absence of growth in thepresence of hygromycin B, as well as by diagnostic PCR using theprimer pair dia_FfCCL1_5′ and TrpC-T in case of F. fujikuroias well as dia_IL_Com_FgCCL1_5F and trpC-T in case ofF. graminearum (Supplementary Figure S3).

Standard Molecular TechniquesFor DNA isolation of both fungi, lyophilized mycelium wasground to a fine powder and resuspended in extraction bufferaccording to Cenis (1992). Isolated DNA was used for PCRamplification and Southern blot analysis. In case of diagnosticPCR, PCR reactions were set up as follows: 25 ng genomic DNA,5 pmol of each primer, 200 nM deoxynucleoside triphosphates,and 1 unit BioThermTM DNA polymerase (GeneCraft GmbH,Lüdinghausen, Germany). Reactions were performed with aninitial denaturating step at 94◦C for 3 min followed by 35 cyclesof 1 min at 94◦C, 1 min at 56–60◦C, 1 min/kb at 70◦C and afinal elongation step at 70◦C for 10 min. Amplification of knock-out, complementation and cross-complementation constructswas accomplished using a proof-reading polymerase. Therefore,PCR reactions contained 25 ng genomic DNA, 5 pmol of eachprimer, and 1 unit of Phusion R© polymerase (Finnzymes, ThermoFisher Scientific, Finland). Plasmid DNA from S. cerevisiaewas extracted using the yeast plasmid isolation kit (SpeedPrep,DualsystemsBioTech) and directly used for PCR reaction. In caseof knock-out mutants, F. fujikuroi and F. graminearum genomicDNA that was subsequently applied for Southern blot analysis.Therefore, genomic DNA was digested with SacI and XbaI,respectively, separated on a 1% (w/v) agarose gel and transferredonto nylon membranes (Nytran R© SPC, Whatman) by downwardblotting (Ausubel et al., 1987). Then, 32P-labeled probes weregenerated using the random oligomer-primer method andhybridized to the membranes overnight at 65◦C according to

the protocol of (Sambrook et al., 1989) and subsequently washedwith 1x SSPE (0.18 M NaCl, 10 mM NaH2PO4 and 1 mMEDTA, pH 7), 0.1% (w/v) SDS at the same temperature. Incase of F. fujikuroi, the downstream region amplified withthe primer pair FfCCL1_3F and FfCCL1_3R was used forprobing (Supplementary Figure S2). For F. graminearum, hphwas used for probing in order to exclude multiple integrationsof both transformed knock-out fragments. The hph cassette wasamplified using the primer pair hph_F and hph_R. RNA wasextracted using TRIzol Reagent (InvitrogenTM) according tothe manufacturer’s instructions. For cDNA synthesis, 1 µg oftotal RNA was DNase I treated (Fermentas, Schwerte, Germany)and reverse transcribed using the oligo (dT) 12–18 primerand SuperScript II Polymerase [Invitrogen (Life Technologies),Darmstadt, Germany] as described by the manufacturer. Forwestern blot analysis, mycelium from 3 days-old strains wasground to a fine powder with liquid nitrogen and proteinswere extracted using a modified TCA (trichloroacetic acid)protocol from Reyes-Dominguez et al. (2010). Briefly, about200 mg per sample was resuspended in 1 mL TCA (12%,v/v) vortexed vigorously and kept on ice for 10 min priorto centrifugation (10 min, 4◦C, 2015 × g). The supernatantwas discarded and the pellet was washed three times in 1 mLaceton followed by centrifugation (5 min, 4◦C, 2015 × g). Thepellet was dried at 65◦C and finally extracted with 500 µLextraction buffer (100 mM Tris pH 8, 50 mM NaCl, 1% SDS,1 mM EDTA pH 8) with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) in a dilution of 1:200. Aftercentrifugation, the protein concentration of the supernatant wasquantified using the BCA protein quantification kit (Pierce,USA). Roughly, 15 µg of proteins were used for SDS-Pageand subsequent western blotting. The membrane was probedwith H3 C-Term (Active Motif AM39163), H3K4me1 (ActiveMotif AM39297), H3K4me2 (Active Motif AM39141) as wellas three different H3K4me3 (Active Motif AM39159, abcamab8580, Millipore MP#07-473) primary antibodies and anti-rabbit (Sigma A0545) HRP conjugated secondary antibody.Chemiluminescence was detected with ClarityTM ECL WesternSubstrate and ChemDocTM XRS (Bio-Rad).

Pathogenicity AssaysF. fujikuroi pathogenicity assays were performed accordingto Wiemann et al. (2010). Briefly, germinated rice seedlings(Oryza sativa sp. japonica cv. Nipponbare) were inoculatedwith the F. fujikuroi wild type strain and a 1ffccl1 mutant.100 ppm GA3 and H2O served as a positive and negativecontrol, respectively. After 7 days at 28◦C, 80% humidity anda 12 h/12 h light–dark cycle, rice plants were screened fortypical bakanae disease symptoms. For quantification of GA3in planta, experiments were performed in quintuplicates andrice plants were combined before extraction. Non-infected riceseedlings served as a negative control. For F. graminearumpathogenicity assays, the USU-Apogee full-dwarf hard red springwheat (Triticum aestivum cv. USU-Apogee; Reg.no CV-840,PI592742) cultivar was used (Bugbee and Koerner, 1997). Twospikelets per wheat head were inoculated with 1,000 conidiaeach during anthesis. Mock ears were inoculated using water

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instead of spore suspension. Five ears per Fusarium strain wereinoculated and afterward, the wheat heads on the living plantswere covered with moistened plastic bags for the first 24 h toprovide high humidity. Incubation conditions were set to 50%humidity and 20◦C during day-/nighttime and 12 h photoperiod.For quantification of DON in planta, harvesting was performed21 days after infection and plant material was combined prior toextraction.

Secondary Metabolite AnalysisFor F. fujikuroi, analysis of all here studied SMs was accomplishedusing the culture fluids of 7 days-old cultures that werefiltered over a 0.45 µm membrane filter (Millex R©, Millipore)and directly used for analysis without further preparation.F. graminearum strains were grown for 2 weeks on PDAagar and subsequently extracted using MeOH/CH2Cl2/EtOAc(1/2/3, v/v), evaporated and resuspended in acetonitrile/H2O(1/1, v/v). In case of F. graminearum, samples were run ona QTrap 5500 LC–MS/MS System (Applied Biosystems, FosterCity, CA, USA) equipped with a TurboIonSpray electrosprayionization (ESI) source and a 1290 Series high performanceliquid chromatography (HPLC) System (Agilent, Waldbronn,Germany). Chromatographic separation was done at 25◦C on aGemini R© C18-column, 150 × 4.6 mm i.d., 5 µm particle size,equipped with a C18 4 mm × 3 mm i.d. security guard cartridge(Phenomenex, Torrance, CA, USA). The chromatographicmethod, chromatographic and mass spectrometric parametersare described elsewhere (Malachová et al., 2014). In case ofF. fujikuroi, BIK biosynthesis was determined as describedelsewhere (Michielse et al., 2014). For FSA, FUS and GA3measurement, the described HPLC coupled to tandem massspectrometry (HPLC–MS/MS) method was transferred to aQTrap 6500 mass spectrometer (AB Sciex, Darmstadt, Germany)(Michielse et al., 2014). Data acquisition was performed withAnalyst 1.6.2 (AB Sciex). Briefly, for FUS and GA3 analysis, 10 µLof the culture filtrate was mixed with 10 µL methyl parabene(MePa, 10 µg/mL) and the solution was diluted with 980 µLwater, resulting in 0.1 µg/mL MePa. For FSA, 10 µL of the culturefiltrate was diluted with 990 µL H2O and 10 µL of this solutionwas mixed with MePa as described above. Chromatographicseparation was carried out on a 150 mm × 2.1 mm i.d., 5 µm,Eclipse XDB-C18 column (Agilent Technologies, Waldbronn,Germany) with methanol + 1% (v/v) formic acid (FA) assolvent A and water + 1% (v/v) FA as solvent B. The columnwas tempered to 40◦C. After isocratic running for 3 min at15% A, the gradient rose up to 100% A in 10 min. Withthe concentration of solvent A, the flow rate also rose from400 to 450 µL/min. After holding these conditions for 2 min,the column was equilibrated for 3 min. A divert valve wasused to discard polar substances from the medium in the first3 min of the run. For MS/MS analysis with ESI, the followingparameters were used: the curtain gas (nitrogen) was set to30 psi, the nebulizer gas (zero-grade air) to 35 psi and thedrying gas (zero-grade air) to 40 psi. The ion spray voltagewas set to +5500 V in the positive and to −4500 V in thenegative mode. Nitrogen was also used as collision gas in mediummode.

FUS was analyzed with a declustering potential (DP) of+160 V, an entrance potential (EP) of +10 V and a collisionenergy (CE) of +27 V. The cell exit potential (CXP) was adaptedfor every transition as described below. Dwell times were setto 20 ms. As parent ion for FUS analysis, the sodium adductof FUS was used with the following transitions: quantifier FUS(M+Na)+: m/z 454.1→290.1 (CXP+26 V), qualifier 1 FUS: m/z454.1→335.1 (CXP +24 V), qualifier 2 FUS: m/z 454.1→267.2(CXP+20 V).

FSA was analyzed with a CXP of +10 V, a DP of +40 V andan EP of +10 V. Dwell times were set to 20 ms. Quantifier FSA(M+H)+: m/z 180.2→134.1 (CE +23 V), qualifier 1 FSA: m/z180.2→91.9 (CE +35 V), qualifier 2 FSA: m/z 180.2→65.1 (CE+49 V).

For MePa and GA3 analysis, negative polarity was used.The DP was set to −90 V for GA3 analysis and −50 V forMePa analysis. The CE and the CXP varied for each transition.Quantifier MePa (M-H)−: m/z 151.0→92.0 (CE −23 V, CXP−11 V), qualifier MePa: m/z 151.0→121.0 (CE −28 V, CXP−11 V). Quantifier GA3 (M-H)−: m/z 345.0→239.1 (CE − V,CXP−19 V), qualifier 1 GA3: m/z 345.0→143.0 (CE−36 V, CXP−13 V), qualifier 2 GA3: m/z 345.0→221.0 (CE −28 V, CXP−11 V). All experiments were performed in triplicates and aStudent’s t-test was done to determine statistical significance.

To quantify SMs in planta, 20 mg of ground rice plants incase of F. fujikuroi and 100 mg of ground wheat ears in caseof F. graminearum were extracted as described in Bueschl et al.(2014). Briefly, samples were extracted with 1 mL extractionsolution containing methanol (1% FA) and 1% FA (3:1, v/v)and then treated with ultrasound for an additional period of15 min. The extract was centrifuged for 10 min at 20817 × gand 4◦C. An aliquot of 700 µL was transferred to a clean tube.After addition of 350 µL 1% FA and agitation, samples weredirectly used for HPLC–MS/MS measurements as previouslydescribed (Malachová et al., 2014). Mock-infected rice and wheatplants served as negative controls. Quantified metabolite levelswere normalized to the input sample weights. For metabolitequantification, the concentration of the desired SMs was relatedto the biomass accumulation; production level of the wild typewas arbitrarily set to 100% for direct comparison.

Chromatin Immunoprecipitation (ChIP)F. fujikuroi strains were grown for 72 h in DVK medium at28◦C. An aliquot of 0.5 mL was then transferred to syntheticICI medium with either 6 or 60 mM glutamine as the solenitrogen source. After an additional growth period of 72 h, themycelium was crosslinked with 1% (v/v) formaldehyde for 15 minat 28◦C and 90 rpm. In case of F. graminearum, the fungalstrains were grown on solid PDA at 20◦C and harvested 4 dayspost-inoculation. For crosslinking, mycelia were transferred toliquid potato dextrose broth containing 1% (v/v) formaldehydeand incubated for 15 min at 20◦C and 90 rpm. Quenchingwas performed in both cases by addition of 125 mM glycine.Mycelia were filtered over miracloth and directly ground inliquid nitrogen. The following antibodies were used in this study:H3K4me2 (Active Motif, AM39141), H3K4me3 (Merck MilliporeMP#07-473) and H3K9ac (Active Motif AM39137). The ChIP

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protocol was adapted from the protocol described elsewhere(Reyes-Dominguez et al., 2012). Sonication was performed in aBioruptor R© Standard sonication device (Diagenode) for 30 min,2 min on/1 min off at maximum power. Precipitation of theprotein-antibody conjugate was performed with Dynabeads R©

Protein A (Novex R©, Life Technologies, 10002D). Washing wasdone applying 3x Wash Buffer and 1x Final Wash Buffer followedby elution in TES buffer (50 mM Tris-HCl pH 8, 10 mM EDTA,1% SDS). Histone-bound DNA was treated with Proteinase K(Thermo Scientific) and DNA purification was done using theMiniElute PCR Purification Kit (Qiagen). Amplification anddetection of precipitated DNA in qRT-PCR was performed withiQTM SYBR R© Green Supermix (Bio-Rad) following the providers’instructions. All experiments were performed in biological andtechnical duplicates.

Reverse Transcriptase-Quantitative PCR(RT-qPCR) and ChIP-coupled qPCRReverse transcriptase-quantitative PCR (RT-qPCR) wasperformed with iQ SYBR Green Supermix (Bio-Rad, Munchen,Germany) using an iCycler iQ Real-Time PCR System (Bio-Rad).To quantify mRNA levels of SM genes, the followingprimers were used: AUR gene cluster (AUR1, Aur1_ORF_fwd//Aur1_ORF_rev; PKS12, RT-PCR_PKS12_F//RT-PCR_PKS12_R), DON gene cluster (TRI5, Tri5_ORF_fwd//Tri5_ORF_rev;TRI6, Tri6_ORF_fwd //Tri6_ORF_rev), ZON gene cluster(PKS13, RT-PCR_PKS13_F//RT-PCR_PKS13_R; PKS4,PKS4-PS.2//PKS4-PS.1). Levels of mRNA were related toconstitutively expressed reference genes, i.e., FGSG_06257encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH)(GAPDH_qPCR_fwd//GAPDH_qPCR_rev), FGSG_07335encoding actin (qPCR_actin_F// qPCR_actin_R) and FGSG_09530 encoding ß-tubulin (cDNA_ß-TUB_F //cDNA_ß-TUB_R). Primer efficiencies in the RT-qPCR were kept between90 and 110%. Relative expression levels were calculated usingthe44Ct method (Pfaffl, 2001). Experiments were performed inbiological and technical duplicates. Primer sequences are listedin Supplementary Table S1.

To reveal changes in the desired histone marks atSM cluster genes, the following primer pairs were usedfor qPCR in case of F. fujikuroi: copalyl diphosphatesynthase/kaurene synthase /KS (FFUJ_14336, qRT-PCR_CPS/KS_F//qRT-PCR_CPS/KS_R), P450 monooxygenase P450-1(FFUJ_14333, qRT-PCR_P450-1_F//qRT-PCR_P450-1_R),P450-2 (FFUJ_14334, qRT-PCR_P450-2_F//qRT-PCR_P450-2_R) and P450-4 (FFUJ_14332, qRT-PCR_P450-4_F//qRT-PCR_P450-4_R) for the GA gene cluster, PKS BIK1 (FFUJ_06742, qRT-PCR_bik1_F//qRT-PCR_bik1_R) and theZn2Cys6 transcription factor (TF) BIK5 (FFUJ_06746, qRT-PCR_bik5_F//qRT-PCR_bik5_R) in case of the BIK gene cluster,PKS – NRPS FUS1 (FFUJ_10058, qRT-PCR_fus1_F//qRT-PCR_fus1_R) and a pathway-specific methyltransferase FUS9(FFUJ_10050, qRT-PCR_fus9_F//qRT-PCR_fus9_R) for theFUS gene cluster. Sequences of the respective ORFs wereextracted from the publicly available genome sequence ofF. fujikuroi (Wiemann et al., 2013). In case of F. graminearum,the GAL4-like Zn2Cys6 TF AUR1 (FGSG_02320) and PKS12

(FGSG_02324) (Frandsen et al., 2006) were analyzed withprimers Aur1_K4me_fwd and Aur1_K4me_rev as well asPks12_K4me_fwd and Pks12_ORF_rev, respectively. In caseof the core DON gene cluster (Desjardins et al., 1993; Hohnet al., 1993; Kimura et al., 2003), the trichodiene synthase(sesquiterpene cyclase) gene TRI5 (FGSG_03537) (Hohn andBeremand, 1989) was analyzed with primers Tri5_K4me_fwd andTri5_K4me_rev; and the TF-encoding TRI6 gene (FGSG_16251)(Proctor et al., 1995; Hohn et al., 1999) was analyzed with primersTri6_K4me_fwd and Tri6_K4me_rev. Relative amounts of DNAwere calculated by dividing the immune-precipitated DNA bythe input DNA. Experiments were performed in duplicates andeach ChIP was done in two biological repetitions.

For determining the F. fujikuroi and F. graminearuminfection rates, total gDNA (of the fungus and the plant)was isolated from infected and lyophilized plant materialusing the NucleoSpin Plant II Kit (Machery Nagel) for theF. fujikuroi infection as well as the DNeasy R© Plant MiniKit (Qiagen) for the F. graminearum infection. Calculationof the infection rate is based on qPCR quantification of theproportion of fungal gDNA within the fungus/plant gDNAmixture (Brunner et al., 2009). Subsequent qPCR analysis wasperformed using iQTM SYBR R© Green Supermix (Bio-Rad) andprimers BIK1_F//BIK1_R for quantification F. fujikuroi gDNA,primers Pks12_ORF_fwd//Pks12_ORF_rev for quantification ofF. graminearum gDNA as well as primers ITS1P//ITS4 forquantification of plant gDNA (wheat or rice).

RESULTS

Identification and Cloning of CCL1 inF. fujikuroi and F. graminearumThe cclA homologs in F. fujikuroi and F. graminearum,FfCCL1 and FgCCL1, respectively, were identified by BLASTpanalysis using the protein sequence of Aspergillus nidulans(AN9399). Clustal W alignment showed 83.6% sequenceidentity of Ccl1 on protein level in both fusaria (SupplementaryFigure S4). To study the functions of FfCCL1 (FFUJ_04433)and FgCCL1 (FGSG_09564), both genes were deleted byhomologous integration of a hygromycin resistance cassetteinto the respective wild type strains IMI58289 (F. fujikuroi)and PH-1 (F. graminearum), hereafter referred to as FfWTand FgWT. Correct integration of the resistance cassette wassubsequently verified by diagnostic PCR and Southern blotanalysis (Supplementary Figures S2B,C). Three independentdeletion mutants each, that is 1ffccl1_T9, 1ffccl1_T10 and1ffccl1_T11 as well as 1fgccl1_T1, 1fgccl1_T3 and 1fgccl1_T4,were obtained for F. fujikuroi and F. graminearum, respectively.All mutants of F. fujikuroi and F. graminearum showedan identical phenotype. Hence, 1ffccl1_T9 and 1fgccl1_T1were arbitrarily chosen for complementation approaches.Complementation was done by homologous re-integrationof the native genes driven by the native promoters into the1ffccl1- and 1fgccl1-mutant backgrounds resulting in threeindependent mutants each for F. fujikuroi (FfCcl1Cil_T2, T7,T14) and F. graminearum (FgCcl1Cil_T6, T7, T8), that showed

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identical phenotypes and correct in loco integration of FfCCL1and FgCCL1, respectively (Supplementary Figure S3A).

Deletion of CCL1 has Only Minor Impacton Fungal DevelopmentTo determine the influence of Ccl1 on fungal development,hyphal growth and conidiation were investigated in both fungi.Hyphal growth was only slightly, but significantly, decreasedin the 1ffccl1 deletion mutant on all selected media, i.e., CM(to about 87%), PDA (to about 80%) and Czapek Dox (CD,

to about 92%), compared to FfWT (Figure 1A). Similarly,deletion of FgCCL1 in F. graminearum resulted in slightlyreduced growth on PDA (to about 93%) and CD (to about75%) compared to FgWT, while no growth defect was observedon CM (Figure 1A). Complementation of 4ffccl1 and 4fgccl1with the native genes FfCCL1 and FgCCl1, respectively, rescuedthe wild type phenotypes (Figure 1A). Regarding conidiaformation, there is a significant difference between both fusaria.In general, F. fujikuroi is able to produce micro- as well asmacroconidia. However, the FfWT strain (F. fujikuroi IMI58289)

FIGURE 1 | Lack of Ccl1 results in slightly reduced hyphal growth and has no impact an asexual development. (A) The wild type strains (Fusariumfujikuroi – FfWT, Fusarium graminearum – FgWT), CCL1 deletion mutants (F. fujikuroi – 4ffccl1, F. graminearum – 4fgccl1) and in loco complementation strains(F. fujikuroi – FfCcl1Cil, F. graminearum – FgCclCil) were grown for 5 days on solid complete medium (CM), PDA and Czapek Dox (CD) medium at 28 and 20◦C incase of F. fujikuroi (left panel) and F. graminearum (right panel), respectively. Experiments were done in triplicates. Mean values and standard deviations are given inthe diagram. Hyphal growth of the wild type strains on the respective media was arbitrarily set to 100%. Asterisks above the bars denote significant differences in themeasurements of the indicated strains compared to the respective wild type, p < 0.001. (B) Conidiation was assessed on vegetable V8 agar in case of F. fujikuroi(left panel) and in case of F. graminearum in liquid carboxymethyl cellulose (right panel). Conidia production of the respective wild type strains was arbitrarily set to100%. Experiments were done in triplicates. Mean values and standard deviations are shown. All experiments were performed with three independent CCL1 deletionand complementation strains all showing an identical phenotype. Hence, only one of each is shown.

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used in this study produces only microconidia in long chains(Wiemann et al., 2013). In contrast, F. graminearum does notproduce microconidia but only slender macroconidia with 5to 6 septa (Leslie and Summerell, 2007). Neither micro- normacroconidia formation was affected by deletion of CCL1 inF. fujikuroi and F. graminearum, respectively (Figure 1B).Taken together, growth behavior was only slightly affected upondeletion of CCL1 in both fungi, while conidiation remainedunchanged.

Reduced Virulence Factor Biosynthesisof Mutants in Axenic Cultures is Rescuedduring PathogenesisSeveral SMs have been described as virulence factors, includingGA and DON production in F. fujikuroi and F. graminearum,

respectively (Jansen et al., 2005; Maier et al., 2006; Wiemann et al.,2013). To analyze if the deletion of CCL1 affects the biosynthesisof the two species-specific virulence factors, the fungal wildtype and mutant strains were grown under their respectiveproduction conditions, i.e., liquid synthetic ICI medium with6 mM glutamine (nitrogen limitation) in case of GA, andPDA in case of DON (Giese et al., 2013; Wiemann et al.,2013). Deletion of CCL1 resulted in a significantly decreasedbiosynthesis of both virulence factors, GA (about 56% of FfWT)in F. fujikuroi and DON (about 47% of FgWT) in F. graminearum.Complementation of the 1ccl1 mutant strains restored thebiosynthesis of both virulence factors to the respective wild typelevels (Figure 2A). Hence, a reduced virulence of the CCL1deletion mutants was expected for both fungi. However, boththe F. fujikuroi wild type and 1ffccl1 mutant strains causedtypical bakanae symptoms on rice seedlings (Figure 2B). No

FIGURE 2 | Wild type-like virulence of 4ffccl1 and 4fgccl1 mutants is accompanied by enhanced virulence factor production in planta compared tothe axenic cultures. (A) The F. fujikuroi wild type (FfWT), 4ffccl1 and FfCcl1Cil (left panel) were grown in liquid ICI medium with 6 mM glutamine for determination ofgibberellic acid (GA3) at 28◦C. Filtrates of 7 day-old liquid cultures were applied for HPLC–MS/MS measurements. In case of F. graminearum (right panel), the wildtype (FgWT), 1fgccl1 and FgCcl1Cil were grown for 2 weeks on PDA at 20◦C for deoxynivalenol (DON) quantification. Agar plugs were subsequently extracted andanalyzed by HPLC–MS/MS. For a direct comparison, production of the wild type was arbitrarily set to 100%. To exclude differences in the biosynthesis due todifferent growth behaviors, product formation was correlated to biomass formation. Experiments were performed in triplicates. Asterisks above the bars denotestatistical significance, p < 0.005. (B) Rice seedlings (left panel) and wheat heads (right panel) were infected with F. fujikuroi FfWT and 4ffccl1 mutant andF. graminearum FgWT and 4fgccl1 mutant. Typical disease symptoms were assessed 7 and 21 days past infection (dpi) in case of F. fujikuroi and F. graminearum,respectively. (C) For in planta GA and DON quantification, five plants were infected each with either FfWT and 4ffccl1 or FgWT and 4fgccl1. Plants were combinedand the total amount of GA3 and DON was extracted and quantified by HPLC–MS/MS. Experiments were performed in biological duplicates; nd, not detectable.

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significant differences regarding shoot elongation and chlorosiscompared to FfWT were observed. Similarly, infection of wheatheads with the F. graminearum 1fgccl1 mutant resulted in aphenotype that was undistinguishable from the wild type strainFgWT (Figure 2B). Both mutant strains showed wild type-likefungal invasion (Supplementary Figure S5). To understand thecorrelation between reduced production of virulence factors inaxenic culture and the wild type-like virulence of both mutantstrains on their respective host plants, production of GA andDON was determined also in planta. Unexpectedly but inagreement with the wild type-like virulence, GA and DON levelswere restored to wild type levels in both mutant strains in planta(Figure 2C).

Ccl1 Function Generates DistinctTranscriptional Readouts Depending onthe SM LocusTo study the role of Ccl1 for the regulation of other SMs,the production of several known metabolites was analyzed inboth fusaria. In F. fujikuroi, the production of the mycotoxinsFUS and FSA, both induced under nitrogen sufficient (60 mMglutamine) conditions (Niehaus et al., 2013, 2014b), as well asproduction of BIK, induced under nitrogen limiting (6 mMglutamine) conditions (Wiemann et al., 2009), was assessed. Incase of F. graminearum, production of the mycotoxins FUS andZON as well as the pigment AUR was determined on PDA asdescribed by Giese et al. (2013). Production yields of SMs weresubsequently analyzed by HPLC or HPLC–MS/MS (Malachováet al., 2014; Michielse et al., 2014). In contrast to the reduced GAand DON levels, several other SMs were either not significantlyaltered (e.g., FSA in F. fujikuroi or AUR in F. graminearum) oreven increased in the CCL1 deletion strains compared to theirrespective wild types (Figures 3A,B). We noted a very strongoverproduction of ZON in the F. graminearum mutant (abouteightfold) as well as of BIK (about 18-fold) and FUS in F. fujikuroi(roughly 4.5-fold). Fusarins are also slightly increased (about 1.4-fold) in the F. graminearum mutant. Complementation of bothmutant strains restored in each case the respective wild typephenotype.

Cross-Complementation Demonstratesthat Ccl1 Function is Conserved in BothFusariaTo analyze if Ccl1 from one fungus is able to complementloss of the respective gene in the other, cross-complementationexperiments were performed by in loco integration of FgCCL1into 1ffccl1 driven by the native F. fujikuroi promoter as well as inloco integration of FfCCL1 driven by the native F. graminearumpromoter into the 1fgccl1 background. Several mutants wereobtained that showed correct in loco integration of FgCCl1 andFfCCL1 in the 1ffccl1 and the 1fgccl1 mutants, respectively(Supplementary Figure S3B). In both cases, three independentcross-complementation mutants were taken for subsequentexperiments. As all of them showed an identical phenotype,only one of each strain, that is 1ffccl1/FgCCL1_T4 and1fgccl1/FfCCL1_T22, is shown. To study if FgCCl1 complements

1ffccl1, and thus, restores SM production to FfWT-level, fungalstrains were cultivated under inducing conditions for each SM(liquid synthetic ICI medium with 60 mM glutamine in case ofFUS and 6 mM glutamine in case of BIK and GA). SM productionwas quantified after 7 days of incubation. The significantlyderegulated SMs in the 4ffccl1 mutant, that is increased FUSand BIK biosynthesis and decreased GA biosynthesis, wererestored to FfWT-level in 1ffccl1/FgCCL1 (Figure 4). In caseof F. graminearum, SM production of the FgWT, 4fgccl1and the cross-complementation strains (1fgccl1/FfCCL1) wasquantified after 2 weeks of fungal growth on PDA. Similarto F. fujikuroi, the deregulated production of SMs in the4fgccl1 mutant, that is increased FUS and ZON biosynthesisand decreased DON biosynthesis, was restored to FgWT-levelin 1fgccl1/FfCCL1 (Figure 4). Taken together, all observedCCL1 deletion phenotypes were restored to the respective wildtype levels by cross-complementation indicating high functionalconservation of Ccl1 in both distantly related fusaria.

Ccl1 is Necessary for Full Global H3K4TrimethylationTo analyze the impact of CCL1 deletion on H3K4 methylationof all nuclear histones in both fusaria, we performed westernblot analysis using H3K4 mono-, di-, and trimethylation-specificantibodies (Figure 5). In both fungi, deletion of CCL1 resulted instrongly reduced, but not abolished, H3K4me3-marked histones.Because antibody specificity is always an issue in chromatinanalysis, we used different antibody sources for H3K4me3 andall gave comparable results. Complementation as well as cross-complementation restored H3K4me3 to wild type levels in bothfungi (Supplementary Figure S6). Thus, similar to the findingsin S. pombe, Arabidopsis thaliana, and Drosophila melanogaster(Jiang et al., 2011; Mohan et al., 2011; Mikheyeva et al.,2014), lack of the bre2 homolog Ccl1 causes great decrease ingenome-wide H3K4me3 but not in H3K4me1/2 levels in bothFusarium spp.

Ccl1 Regulates Balance between H3K4Di- and Trimethylation Independently ofTranscriptional Status of SM GenesNext we analyzed H3K4 methylation levels in more detailat the affected SM clusters using these antibodies to analyzegene-specific H3K4 methylation by qPCR-coupled ChIP. Toidentify possible gene-specific changes in the CCL1 deletionmutants, we tested genes in the FUS, BIK and GA clustersin F. fujikuroi as well as genes in the FUS, ZON and DONclusters in F. graminearum. For this, F. fujikuroi strains werecultivated in synthetic ICI medium under nitrogen starvation(6 mM glutamine for BIK and GA) and nitrogen surplus (60 mMglutamine for FUS) conditions for 3 days, a time point whentranscripts of all investigated SMs are clearly detectable in theFfWT (Wiemann et al., 2009; Niehaus et al., 2013, 2014b). In caseof F. graminearum, ChIP was performed from cultures grownfor 4 days on solid PDA, when transcripts were detectable inthe FgCCL1 deletion strain and differences in SM transcriptionwere highest (Supplementary Figure S7). Importantly, expression

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FIGURE 3 | Deletion of CCL1 results in increased production of some secondary metabolites (SMs) in both fusaria. The F. fujikuroi wild type strain (FfWT),the CCL1 deletion mutant (4ffccl1) and in loco complementation (FfCcl1Cil) were grown for 7 days in synthetic ICI medium with either (A) 60 mM glutamine (nitrogensurplus) for fusarin and fusaric acid production or (B) 6 mM glutamine (nitrogen starvation) for bikaverin production (left panel). Liquid cultures were filtered overmiracloth and subsequently directly applied for HPLC–DAD analysis (bikaverin) or HPLC–MS/MS (fusarins and fusaric acid). To exclude falsifications in SMbiosynthesis due to differences in hyphal growth, SM production was correlated to biomass formation. The F. graminearum wild type strain (FgWT), the 4fgccl1 andthe in loco complementation FgCcl1Cil were grown at 20◦C for 2 weeks on solid PDA for (A) fusarin, zearalenone and (B) aurofusarin production (right panel). Agarplugs were subsequently extracted and applied for HPLC–MS/MS analysis. In each case, three independent mutants were analyzed and all behaved identically, thusonly one is shown. SM production of the respective wild type was arbitrarily set to 100%. Mean values and standard deviations are given. Asterisks above the barsdenote significant differences in the measurements of the indicated strains compared to the respective wild type. p < 0.001.

of all analyzed SM biosynthetic genes is in agreement with theSM levels recorded in the wild type and mutant strains (compareFigure 2 and Supplementary Figure S7).

Primers for gene-specific qPCR after ChIP were designed totarget in each case the first one or two nucleosomes of thecoding region, because it has been shown in numerous organismsincluding F. graminearum (Connolly et al., 2013) and A. nidulans(Gacek-Matthews et al., 2016), that these are the ones mostprominently labeled with H3K4 di- or trimethylation. Although,genome-wide reduction of H3K4me3 in 1fgccl1 and 1ffccl1 wasevident in western blot analysis, no such clear changes wereobserved at the investigated SM cluster genes in either of thetwo Fusarium spp. (Figure 6), with the exception of FUS1 inF. fujikuroi (Figure 6, left panel, first graph). Here, the relativeamount of H3K4me3 in the mutant was reduced to roughly20% compared to the FfWT. All other genes showed either no

or only subtle reduction in H3K4me3 levels in the mutantsconsistent with the observed generally very low levels of thismark in SM gene clusters which cannot be reduced further.To validate the results from the SM gene clusters, we alsoquantified H3K4me3 levels in the actin-encoding gene (ACN)that is known to carry high levels of these marks in bothfusaria. Here, in agreement with the global histone analysis,H3K4me3 levels were significantly decreased to about 53 and54% in case of F. fujikuroi and F. graminearum, respectively(Supplementary Figure S8). At these loci, we detected for thefirst time an interdependency between H3K4 methylation marksas concomitantly with decreased H3K4me3 levels, the amountof H3K4me2 was increased in the CCL1 deletion strain ofF. fujikuroi, and slightly also in F. graminearum.

In the latter pathogen, the investigated SM cluster genes hadno change in H3K4 dimethylation and the results were consistent

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FIGURE 4 | Ccl1 shows high functional conservation in both fusaria. For secondary metabolite (SM) production in case of Fusarium fujikuroi (Left), theF. fujikuroi wild type (FfWT), CCL1 deletion (4ffccl1) and cross-complementation strains (4ffccl1/FgCCL1) were grown in liquid synthetic ICI medium with either60 mM (fusarin) or 6 mM (bikaverin and gibberellic acid) glutamine at 28◦C. Liquid cultures were filtered over miracloth and directly used for HPLC–DAD (in case ofbikaverin) or HPLC–MS/MS (in case of fusarins and gibberellic acid) analysis. To exclude differences in the biosynthesis due to different growth behaviors, productformation was correlated to the biomass. In case of Fusarium graminearum (Right), the wild type (FgWT), 4fgccl1 and 4fgccl1/FfCCL1 strains were cultivated for2 weeks on PDA at 20◦C. Subsequently, agar plugs were extracted and applied for HPLC–MS/MS analysis. Experiments were performed in triplicates with at leastthree independent mutants. SM production of the respective wild type strains was arbitrarily set to 100%. Mean values and standard deviations are given. Asterisksabove the bars denote significant differences in the measurements of the indicated strains compared to the respective wild type. p < 0.005.

in all investigated genes. In contrast, two out of three SM geneclusters in F. fujikuroi showed a strong switch between bothH3K4me forms in the mutant. Genes in the investigated BIK aswell as GA cluster showed an enormous enrichment of H3K4me2at their 5′-end nucleosomes (Figure 6, left panel). It is importantto note, that these two gene clusters are the ones investigatedhere which are positioned in typical subtelomeric regions that areknown to be highly decorated with this mark and influenced byBre2/CclA during subtelomeric gene silencing (Miller et al., 2001;Krogan et al., 2002; Bok et al., 2009; Mikheyeva et al., 2014).

In a previous study employing ChIP-seq in the FfWT,we found out of all analyzed SM gene clusters inducedunder nitrogen-limiting conditions significant H3K4me2 levelsin only two genes of the GA cluster (P450-2 and P450-4)that are (Wiemann et al., 2013). To analyze if the observedchanges in dimethylation levels can also be seen at these two

genes, H3K4me2 levels were additionally quantified for P450-2 and P450-4. Similar to CPS/KS and P450-1, H3K4me2 levelswere also significantly increased at the two P450 genes upondeletion of FfCCL1 (17-fold at P450-2 and 12-fold at P450-4)(Supplementary Figure S9). Notably, enrichments in H3K4me2were comparable to H3K4me2 levels at ACN (SupplementaryFigure S10). However, expression of biosynthetic activities didnot necessarily correlate with this strong H3K4me2 decorationas in the case of bikaverin, the genes carrying high H3K4me2 aremore active in the mutant whereas the GA cluster is less active inthe mutant despite the strongly increased H3K4me2 levels.

As gene expression of several SM clusters was alteredprofoundly, we also examined the H3K9 acetylation status(H3K9ac) in wild type and CCL1 mutant strains. Previously, wehave shown that H3K9ac is associated with active transcriptionof several SM gene clusters in F. fujikuroi, including the ones

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FIGURE 5 | Lack of Ccl1 results in decreased global H3K4me3. The Fusarium wild type strains (F. fujikuroi – FfWT, F. graminearum – FgWT) and the respectiveCCL1 deletion mutants (F. fujikuroi – 1ffccl1, F. graminearum – 1fgccl1) were grown for 3 days on solid complete medium. Whole protein extracts were subsequentlyisolated from lyophilized mycelia and roughly 15 µg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used fordetection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.

investigated here (Niehaus et al., 2013; Studt et al., 2013). Wefound that H3K9ac levels were significantly increased in the1ffccl1 mutant at the FUS (about 1.9-fold for FUS1 and 4.1-fold for FUS9) and the BIK cluster genes (4.1-fold for BIK1and 4.2-fold for BIK5) (Figure 6), which is in agreementwith their elevated expression activities. However, also the GAcluster showed significant enrichment of H3K9ac in the FfCCL1deletion strain (fivefold at CPS/KS, threefold at P450-1, threefoldcompared to FfWT) (Figure 6), albeit a reduced GA productionlevel was recorded under these conditions (Figure 3A). In caseof F. graminearum, a slight yet statistically significant increasein H3K9ac was seen only for some of the investigated SM genesupon deletion of CCL1, but it is questionable if these small effectsare biologically relevant.

DISCUSSION

In close proximity to promoters, H3K4me3 is thought to promotethe transition of RNA polymerase II (Pol II) to active elongation(Ardehali et al., 2011). Methylation of H3K4 is mediatedby the methyltransferase Set1 that catalyzes mono-, di- andtrimethylation and is itself integrated in the COMPASS complex(Roguev et al., 2001; Krogan et al., 2002; Schneider et al., 2005).In contrast to animals and yeasts, where the roles of Set1 andits complex partners are firmly established in transcriptionalcontrol and genome stability (Shilatifard, 2012), the functionof Set1 was studied just in a few cases in the filamentousascomycetes (pezizomycotina). In Neurospora crassa Set1 is notessential and has been shown to regulate the circadian rhythm

(Raduwan et al., 2013). Also in A. nidulans set11 alone is viablebut synthetic lethal with regulators of mitosis which providesa link between H3K4 methylation and H3S10 phosphorylation(Govindaraghavan et al., 2014). Cells of the rice pathogenM. oryzae lacking MoSet1 cannot develop proper infection andasexual reproduction structures (Pham et al., 2015). In contrastto the quite mild phenotypes of Set1 inactivation mutants inthe species mentioned above, lack of this gene resulted in severegrowth defects, increased stress sensitivity and reduced virulencein F. graminearum (Liu et al., 2015) as well as crippled growth andan almost abolished asexual reproduction in F. fujikuroi. Takentogether, this indicates that H3K4 methylation is an essentialchromatin modification in fusaria required for a number ofcellular processes whereas in N. crassa and A. nidulans thishistone mark seems to play only a role in some specializedprocesses.

Ccl1 is Largely Dispensable for NormalFungal Growth and DevelopmentWhile lack of Set1, the catalytic subunit of COMPASS, hasdrastic effects in both fusaria, removal of the putative adaptorsubunit by deletion of CCL1 had only minor impacts on hyphalgrowth and conidiation in these two fungi. Western blot analysisshowed that in this mutant only H3K4 trimethylation levelsare reduced, whereas H3K4 mono- and dimethylation remainbasically unchanged within the limits of detection by this method.This suggests that mono- and dimethylation are importantfeatures of chromatin in these two species but the observedgenome-wide decrease in H3K4me3 does not drastically alter

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FIGURE 6 | Chromatin immunoprecipitation (ChIP) reveals distinctalterations in H3K4 dimethylation at SM gene clusters. The F. fujikuroiwild type strain (FfWT) and the CCL1 deletion mutant (1ffccl1) were grown insynthetic ICI medium with either 60 mM (fusarin) or 6 mM glutamine (bikaverinand gibberellic acid) for 3 days. Mycelium was crosslinked and used for ChIPanalyses (Left). In case of F. graminearum, the wild type (FgWT) and 1fgccl1were grown for 4 days on PDA (Right). ChIP assays were conducted by usingH3K9 acetylation (H3K9ac)-, H3K4me2- and H3K4me3-specific antibodies.Precipitated genomic DNA was quantified by quantitative real-time PCR usingprimer pairs located in the 5′ region of investigated SM cluster genes. Foreach SM cluster, two cluster genes were analyzed. Experiments wereperformed in biological and technical duplicates. The amount of precipitatedDNA in the respective wild type strain was arbitrarily set to 1. Mean values andstandard deviations are shown. PTMs, post-translational modifications.

genome function and essential transcriptional networks. Again,this is contrary to findings in Aspergillus spp., where lack of theCCL1 homolog cclA in A. nidulans and Aspergillus fumigatusresulted in reduced hyphal growth on various media (Boket al., 2009; Giles et al., 2011; Palmer et al., 2013). A similargrowth defect has been observed in A. nidulans set1 and swd1deletions indicating that the integrity of the COMPASS complexis important for full vigor of this fungus. In fact, recent ChIP-seqanalysis of A. nidulans cells also provided compelling evidencethat basic metabolism genes are highly enriched for H3K4me3

(Gacek-Matthews et al., 2016), and thus loss of this markcan explain the pleiotropic effects on growth and developmentobserved upon deletion of cclA in this fungus.

One possible explanation for the relatively mild effect inCCL1 deletion strains of both fusaria would be that, in contrastto Aspergillus spp. (Palmer et al., 2013) mammals, and yeast(Schlichter and Cairns, 2005; Schneider et al., 2005; Dehé et al.,2006; Dou et al., 2006), deletion of CCL1 in our Fusarium speciesdid not cause any detectable changes in H3K4me2 and someH3K4me3 marks were still present. Notably, H3K4me3 levels atthe actin-encoding genes were reduced though not abolished,suggesting that genes crucial for fungal growth and developmentkeep sufficient levels of H3K4me3 even in the absence of CCL1.

Ccl1 Alters Secondary MetabolismIndependently of H3K4 Methylation atSM LociApart from the slight differences in the methylation pattern inF. fujikuroi and F. graminearum CCL1 deletion compared toAspergillus spp., there is also a similarity between both generaas secondary metabolism was significantly altered upon deletionof CCL1. In both we find up-regulation of SMs produced bygenes localized near the telomeres in the respective fungi. Inthese genomic regions H3K4me3 and several subunits of theCOMPASS complex have been shown to mediate gene silencingalthough the exact mechanism of this process has not beenelucidated yet (Nislow et al., 1997; Miller et al., 2001; Kroganet al., 2002; Sims et al., 2003; Schneider et al., 2005; Woodet al., 2005; Venkatasubrahmanyam et al., 2007; Bok et al.,2009; Margaritis et al., 2012). Going in line with this, lack ofcclA resulted in the induction of monodictyphenone/emodinand the two antiosteoporosis polyketides F9775A and F9775Bin A. nidulans, increased gliotoxin production in A. fumigatusas well as induction of the sesquiterpenoid astellolides inA. oryzae (Bok et al., 2009; Palmer et al., 2013; Shinohara et al.,2016). Strikingly, both non-reducing PKS-encoding genes, mpdG(AN0150) and orsA (AN7909), involved in the biosynthesis ofmonodictyphenone/emodin and F9775A/F9775B, respectively,are localized in subtelomeric regions in A. nidulans (Klejnstrupet al., 2012). Notably, lack of the KDM5-family histonedemethylase KdmB, involved in the removal of H3K4 di- andtrimethylation marks, showed an identical phenotype concerningthese two polyketides (Gacek-Matthews et al., 2016), suggestingthat KdmB targeting H3K4 methylation plays a role in negativeregulation of these clusters in some way. Similarly, several ofthe confirmed and predicted SM gene clusters in F. fujikuroi,including GA and bikaverin, are located in subtelomeric regions(Wiemann et al., 2013). In F. graminearum, predicted SMgene clusters are largely localized in regions enriched forH3K27me3, including subtelomeric but also non-subtelomerichigh diversity regions, that likely result from chromosomefusions reflecting ancestral telomeres and subtelomeric regions(Cuomo et al., 2007; Connolly et al., 2013). Thus, it is temptingto speculate that Ccl1 functions in silencing of SM geneexpression via regulating H3K4 trimethylation also in these twofungi.

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In A. nidulans, increase in SM production was accompaniedby reduced H3K4 di- and trimethylation levels at selectedmonodictyphenone cluster genes and the authors concluded thathigh levels of these marks hinder gene expression (Bok et al.,2009). However, recent genome-wide ChIP-seq experimentsof A. nidulans and F. graminearum revealed that, with fewexceptions, most of the SM cluster genes are poorly decoratedwith these activating marks even under inducing condition(Connolly et al., 2013; Gacek-Matthews et al., 2016). Inaccordance with this, only few SM cluster genes harbor H3K4me2in F. fujikuroi, including P450-2 and P450-4 of the GA cluster(Wiemann et al., 2013), and unchanged H3K4me3 levels atthe investigated SM gene clusters in the 1ccl1 mutant suggestthat this mark is also not abundant at SM gene clusters inF. fujikuroi. Thus, it is questionable if Ccl1 functions directlyin SM gene repression at subtelomeric regions by positioningH3K4 trimethylation at these loci. In fact, as long as theexact mechanism of subtelomeric gene silencing has not beendeciphered, we consider it more likely that Ccl1 rather actsindirectly by regulating additional cis-/trans-acting factors ontranscription of affected SM gene clusters. This interpretation isfurther underlined by observations from this study in which wefound no direct correlation between CCL1 disruption, H3K4me3and the effect on transcriptional activity of SM biosynthetic genes.

Similarly, in M. oryzae where the methyltransferase MoSet1plays a role in gene activation as well as gene repression, nosignificant changes in H3K4 di- and trimethylation at MoSet1-repressed gene groups were found (Pham et al., 2015). Thus,also in this pathogen MoSet1-dependency in the downregulatedgenes do not seem to be a direct consequence of changes inthe H3K4 methylation pattern at the respective gene loci, butrather a result of indirect genetic events. A similar scenario can beimagined for the observed SM phenotypes in the 4ccl1 mutants,in which de-repression of SM genes and concomitant increasein SM production could be due to the insufficient activation ofa repressor protein. Overall, transcriptional regulation by H3K4methylation appears to be different between subtelomeric regionsand the body of chromosomes and only future studies directlytargeting the localization of COMPASS subunits by ChIP will beable to discern direct from indirect effects of regulation by theseimportant transcriptional modulators.

Reduced Virulence Factor Production isRescued during PathogenesisA very interesting aspect of this study was the finding thatpathogenic conditions could reconstitute the very low production

of GA and DON in CCL1 mutants grown under axenic conditionsin shake flasks. The wild type-like virulence of the CCL1 mutantsis probably also the results of this reconstitution effect since bothGA and DON are main virulence factors in F. fujikuroi andF. graminearum, respectively. These findings suggest that a sofar unknown plant-derived signal can compensate for the lackof Ccl1 in planta and remediates virulence factor production.A similar phenotype has been observed for a mutant lacking aglobal activator of secondary metabolism, FfSge1, in F. fujikuroi.Here, GA production was nearly abolished in axenic culture,while GA production was increased up to 50% of the wildtype level during pathogenesis (Michielse et al., 2014). Thesefindings indicate that different regulatory mechanisms operatein planta compared to axenic cultivations, and it is temptingto hypothesize that plant-derived signals not present in axeniccultures function in the pathogen at the level of chromatin andtrigger the production of SMs which are used then by the fungi tosuppress plant defense.

AUTHOR CONTRIBUTIONS

LS, BT, and JS contributed to the design of the work; LS, SJ, BA,MS, and SB were involved in data acquisition; LS, SJ, BT, JS, BA,H-UH, and MS were involved in data analysis; LS and JS wrote themanuscript. All authors revised and approved the manuscript.

FUNDING

We thank the DFG (project TU 101/16-3 and project HU 730/9-3), the Special Research Area ‘SFB’ of Austrian Science (project F-3703), and the NRW Graduate School of Chemistry for financialsupport.

ACKNOWLEDGMENT

We thank Pia Spörhase for wheat cultivations of the USU-Apogeeand Harald Berger for critical reading of the manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.02144/full#supplementary-material

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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